Detection of Salmonella enterica serovar Typhi by nested PCR using different procedures of nucleic acid extraction

Advisor Name:

Choudhury, Prof. NaiyyumQadri, Dr. Firdausi

Department Name:

Department of Mathematical and Natural Science, BRAC University

Publisher

BRAC University

Date Issued:

2010-05

Abstract:

Typhoid fever is one of the major health problems in Bangladesh. It is caused by the
bacterium Salmonella enterica serover Typhi (S. Typhi). The infection rate of S. Typhi is
higher in children. The conventional diagnostic methods of typhoid have limitations.
Most commonly used Widal test gives a high rate of false positive results. As such
PCR method was tried to diagnose typhoid fever in some selected patients of
Bangladesh. Fifty three (53) patients were enrolled in this study from Dhaka hospital
of ICDDR, B and Kamalapur field site. Among them 27 (51 %) persons are male.
Blood was collected from all patients. Three different methods namely, a method by
Haque et al, direct boiling method and commercial Qiagen kit method were tried to
extract Salmonella Typhi DNA from blood and invitro samples. To optimize the DNA
extraction from blood invitro grown S. Typhi bacteria was spiked into blood. From
the three different methods, commercial Qiagen kit method did well comparably to
extract DNA from spiked blood than the other methods. However, none of these
methods did appear promising to extract DNA from patient's blood. Because low
percentage of PCR positive were found from patient's blood culture positive samples.
So another method was tried which was described earlier by Boom's et al. [1990] for
detection of S. Typhi from blood of suspected typhoid fever patient. This method
(Boom's et al.[1990]) showed comparably satisfactory results than the other methods
to deal with patient's blood. In this method the percentage of PCR positive was 35.7
% among the blood culture confirmed patients. DNA extraction was also done in
blood culture negative samples by this method. Here 30.8% positive for PCR was
found among the culture negative samples. However there are some drawbacks in
DNA extraction procedures especially in low concentrated samples. In the method by
Haque et al. [20011 unexpected band was found and same phenomenon was observed
in direct boiling method. This might be due to contamination with S. Typhi. It seems
that, S. Typhi can be detected from patient's blood specimen by PCR method for
diagnostic purpose. However, more study is needed to evaluate the efficacy of PCR
method with some modifications for detecting S. Typhi at low concentration in blood.

Description:

This thesis report is submitted in partial fulfillment of the requirement for the degree of Masters of Science in Biotechnology, 2010

Cataloged from PDF version of thesis report.

Includes bibliographical references (page 33-41).

Keywords

Biotechnology;

Type

Thesis

Copyright

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